Gene transfer composition and method

ABSTRACT

The invention relates to cell culture media more particularly to fertilisation media, to making and using transgenes, to providing sperm cells for fertilisation particularly in applications such as sperm-mediated gene transfer and to using sperm cells for generating transgenic animals.

FIELD OF THE INVENTION

The invention relates to cell culture media more particularly tofertilisation media, to making and using transgenes, to providing spermcells for fertilisation particularly in applications such assperm-mediated gene transfer and to using sperm cells for generatingtransgenic animals.

BACKGROUND OF THE INVENTION

Selective animal breeding is a conventional approach for genotypemodification or improvement. The objective of selective breeding is toincrease the frequency of desirable traits and to decrease the frequencyof less desirable traits in a population. As this approach to genotypemodification is based on the selection of existing genes rather than onthe creation of new genes, it is both time consuming and costly.Further, as new genes are not created by selective breeding, there is alimitation as to the diversity of phenotypes that can be produced byselective animal breeding.

Transgenesis is a more recent approach to genotype modification.Transgenesis is a process by which a nucleic acid molecule, such as agene, that is exogenous, or in other words, foreign to an animal, isintroduced into a genome so that the nucleic acid molecule integratesinto the germ line of the animal and is inherited in a Mendelian manner.An exogenous or foreign nucleic acid molecule that integrates into thegerm line is known as a “transgene”. As genotype modification is basedon the creation of new genes, one consequence of transgenesis is thatboth the time and cost for achieving a desirable trait in a populationare reduced. However, more importantly, the diversity of phenotypes thatcan be achieved by transgenesis is far greater than the diversity thatis achieved by selective animal breeding. This means that animals can begenerated comprising one or more traits that are not common to thespecies from which the animal is derived. For example, a non humananimal can be generated that contains one or more human traits. This hasobvious implications for the uses to which such animals may be put.

To date, the production of transgenic animals has relied almostexclusively on pronuclear microinjection, whereby an exogenous nucleicacid molecule for use as a transgene is physically microinjected intopronuclei of a fertilized ovum. While the technique has been usedsuccessfully to generate transgenic animals in a wide variety ofspecies, the technique has poor efficiency for generating transgenicanimals in species other than mice. More particularly, the frequency offarm animals, such as cattle and pigs, that contain a germ linemodification as a result of pronuclear microinjection is about 0.5 to 4%[Niemann et al. 2000 Anim. Reprod. Sci. 60-61, 277-293]. It has beensuggested that the low efficiency of pronuclear microinjection inanimals other than mice is attributable to factors such as low transgeneintegration rates, unpredictable transgene behaviour and high mortalityrate of manipulated ova [Horan et al. 1991 Arch. Androl 26:83-92; Wall2002 Theriogenology 57:189-201].

Sperm-mediated gene transfer (SMGT) is one of a number of alternativeapproaches for the generation of transgenic animals [Lavitrano et al.1989 Cell 57:717-723; Wall et al. 2002 Theriogenology 57:189-201].According to this approach, an exogenous nucleic acid molecule for useas a transgene is introduced into a sperm cell to transfect the spermcell, and the transfected sperm cell is then used as a vector to deliverthe exogenous nucleic acid molecule to an ovum to provide a transgenicanimal. Some key advantages of SMGT over other transgenic approaches arethat SMGT does not require expensive equipment such as micro-injectorsor micro-manipulators and specialist technical manipulation such assuperovulation and collection and injection of zygotes is not required.According to the SMGT technique, transfected sperm can be used as avector to deliver an exogenous nucleic acid molecule for use as atransgene to an ovum using standard artificial insemination proceduresor by using standard in vitro fertilization procedures.

While SMGT has been used to generate transgenic animals in a widevariety of species, in some species such as pig, the efficiency oftransgenesis has been poor. For example when applied to pigs, thefrequency of offspring in which an exogenous nucleic acid molecule couldbe detected was about 5% [Sperandio et al. 1996 Animal Biotech 7:58-77].None of these animals were demonstrated to contain a transgene; i.e.none of these animals were demonstrated to contain a germ lineintegration of an exogenous nucleic acid molecule. Accordingly, theefficiency of SMGT for production of transgenic pigs is equivalent tothat of other techniques such as pronuclear microinjection. This meansthat SMGT has not yet been demonstrated to provide an improvedefficiency over other techniques for producing transgenic pigs.

It has been proposed that the sequence of an exogenous nucleic acidmolecule for use as a transgene affects the efficiency of SMGT forproduction of transgenic animals such as pigs and cattle [Sperandio etal. 1996 Animal Biotech. 7:58-77]. The objective of the study ofSperandio et al. 1996 was to adopt SMGT protocols originally developedfor murine epididymal cells to use with ejaculated bovine and swinesperm cells. The study found that exogenous nucleic acid molecules foruse as transgenes were substantially modified, most likely byrearrangement.

There is confusion as to the influence of sperm motility on theefficiency of SMGT. While a first study showed a relationship betweenthe motility of bovine sperm cells and an ability of the cells toassociate DNA, no evidence of sperm cell transfection was observed[Castro et al. 1991 Theriogenology 34:1099-1110]. A later study observedthat both motile and damaged, immotile sperm are capable of binding anucleic acid molecule to the sperm plasma membrane [Atkinson et al. 1991Mol. Reprod. And Develop. 29:1-5]. In another study, no rigid conclusioncould be drawn as to the relationship between sperm motility and bindingof a sperm cell to a nucleic acid molecule [Horan et al. 1991 Arch.Androl 26:83-92].

In many circumstances the “ultimate” transgenic animal that is desiredis one that comprises a number of traits that are not inherent in thespecies from which the animal is derived. For example, as it is believedthat many gene products are involved in xenograft rejection, there is aneed to generate a pig comprising tissues that express certain humanantigens and that do not express certain pig antigens. In suchcircumstances, it is particularly important that transgenic animals aremade available in significant numbers for use as founders for providingthe multiple germ line modifications required for the generation of theultimate animal. One way of providing significant numbers of transgenicanimals for use as founders is to increase the numbers of animals foruse in a transgenic program for generating founders. This would increasethe time and cost of such programs.

Optimisation of the efficiency of SMGT would be a better approach. Withoptimised efficiency, the frequency of transgenic animals in progenywould be increased and accordingly, more transgenic animals would bemade available for use as founders to provide the multiple germ linemodifications required for generation of an ultimate animal comprisingthe desired genotype. Accordingly, an advantage of optimised efficiencyof SMGT would be that an ultimate transgenic animal comprising thedesired genotype would be generated more rapidly and with less expense.

SUMMARY OF THE INVENTION

The invention seeks to optimise the efficiency of SMGT for producing atransgenic animal. In a first aspect, the invention provides a mediumfor supporting the viability of a sperm cell. The medium comprises, inwater, glucose in a concentration of about 56 to 69 mM, sodium citratein a concentration of about 31 to 37 mM, EDTA in a concentration ofabout 11 to 14 mM, citric acid in a concentration of about 14 to 17 mMand Trizma base in a concentration of about 48 to 59 mM. Typically themedium has an osmolarity of from about 200 to 320 mOs. Typically themedium has a pH of about 7.4.

The inventors have sought to improve the efficiency of SMGT forproducing transgenic animals, especially for producing transgenic pigs.The inventors have found that the constitution of the medium in whichsperm cells are processed during the SMGT procedure is particularlyimportant for improving the frequency of embryos comprising an exogenousnucleic acid molecule for use as a transgene. They have also found thatthe medium is particularly important for improving the efficiency ofSMGT for producing transgenic animals.

It is surprising that a medium according to the first aspect is capableof improving the efficiency of SMGT because the components of thismedium are unlike those in conventional fertilisation medium, such asTALP [Ball et al. 1983 Biol. Reprod. 28:717-725] and FM [Whittingham1971 J. Reprod. Fert. Suppl. 14:7-21], some of which have been used forSMGT [Sperandio et al. 1996 Animal Biotech. 7:58-77]. As describedherein, when the medium of the invention is used in SMGT for generatingtransgenic embryos, the frequency of embryos comprising an exogenousnucleic acid molecule for use as a transgene is routinely observed to be100%. In view of this improved frequency, one would anticipate animproved frequency of transgenic individuals. Indeed, in experimentsdescribed herein, the frequency of transgenic animals is observed to bein the range of 50 to 60%.

In a second aspect, the invention provides a process for the productionof a medium for supporting the viability of a sperm cell comprisingcontacting glucose in an amount of about 10.1 to 12.4 g, sodium citrate(2H₂O) in an amount of about 9.0 to 11.0 g, EDTA (2H₂O) in an amount ofabout 4.2 to 5.2 g, citric acid (H₂O) in an amount of about 2.9 to 3.6 gand Trizina base in an amount of about 5.9 to 7.2 g, with about 1 litreof water, to form a solution with a pH of about pH7.4 and an osmolarityranging from about 200 to 320 mOs.

In a third aspect, the invention provides a medium for supporting theviability of a sperm cell. The medium is produced by the processaccording to the second aspect of the invention.

In a fourth aspect, the invention provides a composition for providing amedium for supporting the viability of a sperm cell. The compositioncomprises glucose, sodium citrate, citric acid, EDTA and Trizma base inamounts sufficient for providing an aqueous solution having aconcentration of glucose of from about 56 to 69 mM, a concentration ofsodium citrate of from about 31 to 37 mM, a concentration of EDTA offrom about 11 to 14 mM, a concentration of citric acid of from about 14to 17 mM and a concentration of Trizma base of from about 48 to 59 mM.

In another aspect, the invention provides a method for collecting spermcells from an animal for use in sperm-mediated gene transfer. The methodcomprises contacting a sample of semen derived from the animal with amedium according to the first aspect of the invention, to dilute thesample of semen.

In a fifth aspect, the invention provides a method for preparing a spermcell for use in sperm-mediated gene transfer. The method compriseswashing a sperm cell in a medium according to the first aspect of theinvention, to remove seminal fluid from the sperm cell. The sperm cellis washed in conditions for supporting the viability of the sperm cell.

In a sixth aspect, the invention provides a method for transfecting asperm cell with a nucleic acid molecule. The method comprisingcontacting the sperm cell with the nucleic acid molecule in a mediumaccording to the first aspect of the invention. The sperm cell iscontacted with the nucleic acid molecule in conditions for supportingthe viability of the sperm cell.

In a seventh aspect, the invention provides a sperm cell transfectedaccording to the method of the sixth aspect of the invention.

In an eighth aspect, the invention provides a cell, tissue or non-humananimal prepared by fertilisation of an ovum with a sperm cell accordingto the seventh aspect of the invention.

The inventors have also found a direct relationship between sperm cellmotility and the capacity of sperm cells to uptake a nucleic acidmolecule. As described herein, the inventors have found that where the %of motile sperm cells in a sample is less than about 65-70%, the % ofsperm cells in the sample that uptake a nucleic acid molecule is about33% or less. Further, twice the number of sperm cells uptake a nucleicacid molecule in a sample in which 70% or more of sperm are motilesperm, as compared with a sample in which less than 65% of sperm aremotile. This finding has important implications for the efficiency ofSMGT for transgenesis.

Thus, in another aspect, the invention provides a method for determiningwhether a sample of sperm cells is optimal for transfection. The methodcomprises determining whether at least about 65% of the sperm cells inthe sample are motile.

In another aspect, the invention provides a method for selecting asample of sperm cells for transfection. The method comprises:

-   -   (a) determining the motility of sperm cells in a sample; and    -   (b) selecting a sample in which the motility of sperm cells is        determined to be at least about 65%.

In another aspect, the invention provides a method for determiningwhether a sample of sperm cells are optimal for introducing a transgeneinto an oocyte. The method comprises determining whether at least about65% of sperm cells in the sample are motile.

In another aspect, the invention provides a method for selecting asample of sperm cells that are optimised for introducing a transgeneinto an oocyte. The method comprises:

-   -   (a) determining the motility of sperm cells in a sample; and    -   (b) selecting a sample in which the motility of sperm cells is        determined to be at least about 65%.

As described above, the inventors have sought to improve the efficiencyof SMGT for the purpose of increasing the availability of trangenicanimals for use as founders for breeding an “ultimate” animal comprisinga desired genotype. The advantages of this approach include minimisationof time and expenditure for obtaining the desired genotype. Theinventors have also found that these advantages can be obtained by usingSMGT to produce as a founder, an animal that contains more than one typeof transgene.

More specifically, the inventors have found that any one sperm cell canbe transfected with more than one type of exogenous nucleic acidmolecule and that each exogenous nucleic acid molecule can then betransferred to an ovum by the transfected sperm cell according to SMGTto provide a transgenic embryo, from which will develop an individualcomprising more than one type of transgene. In experiments describedherein, the inventors have transfected sperm cells with a cocktail ofexogenous nucleic acid molecules and have observed that all embryosderived from fertilization of ova with these sperm cells comprise thegene products of each exogenous nucleic acid molecule. The finding issurprising because previous studies have shown that transgenes aretypically rearranged after integration and accordingly, are probablyunable to express the desired gene product [Sperandio et al. 1996 AnimalBiotech. 7:58-77]. The finding is of significance because it means thatmany of the intercrosses of particular founders are not required toarrive at an “ultimate” animal comprising the desired genotype.

Thus, in another aspect, the invention provides a method of producing anon-human animal comprising two or more transgenes. The method comprisesthe following steps:

-   -   (a) contacting a sperm cell with two or more exogenous nucleic        acid molecules, each for use as a transgene, to transfect the        sperm cell with each of the two or more exogenous nucleic acid        molecules;    -   (b) fertilising an ovum with the transfected sperm cell to        permit each of the two or more exogenous nucleic acid molecules        to be transferred to the ovum; and    -   (c) maintaining the fertilised ovum in conditions for permitting        the fertilised ovum to form the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the time course of uptake of linear ³H-deoxy-cytidinelabelled EBFP (enhanced blue fluorescent protein) plasmid DNA by pigsperm from 5 boars;

FIG. 2 (i) a blastocyst expressing the three colours (a) blue, (b) greenand (c) red, using filters specific for detection of each fluorescentprotein; (d): white light microscope picture of the blastocyst (×75magnification) and (e): composite picture of colour expression in allthree filters. (ii) shows a negative control i.e. an example of ablastocyst which does not express any of the three colours: (a) whitelight microscope picture of the blastocyst at low power (×10); (b), (c)and (d): show lack of expression of colour in the blue, green and redfilters respectively, (e): composite picture showing lack of colourexpression in all three filters.

FIG. 3 shows the same blastocyst as in FIG. 2 but without thesuperimposing of the 3 colours: (i)(a): white light microscope pictureof the blastocyst (×75 magnification); (b), (c) and (d): expression inthe blue, green and red filters respectively. (ii) shows an example of ablastocyst which does not express any of the three colours: (a) whitelight microscope picture of the blastocyst at low power (×10); (b), (c),and (d) show lack of expression of colour in the blue, green and redfilters respectively.

FIG. 4 shows DNA uptake in pig ejaculated sperm cells. (A) Time courseuptake of end-labeled RSVhDAF plasmid in pig ejaculated sperm cells andnuclear internalization from a selected board. End-labeled RSVhDAFplasmid was incubated with pig ejaculated sperm cells (▴), or pigejaculated and wash sperm cells (▪) at 17° C. as described in Matherialsand Methods. Samples containing about one million sperm were withdrawnat the indicated times and washed thoroughly with SFM. Nuclei wereprepared at each time point from ejaculated and washed sperm cellspre-incubated with labeled DNA (●) as described in Matherials andMethods. Determination of uptake (in cpm) at each point of the timecourse was performing counting 1.106 sperm cells or an equivalent numberof isolated nuclei. Cell or nuclear pellets were dissolved in 100 μl 1MNaOH and incubated for 1 h at 37° C., neutralized with 100μ 1M HCl andcounted in a scintillation counter. (B) Inhibition of DNA uptake by pigejaculated and washed sperm cells in the presence of increasing amountsof seminal fluid. Ejaculated and washed sperm cells (1×10⁶/0.2 ml) frompig z were incubated with the indicated volumes of cell-free seminalfluid for 30 min at 17° C. in SFM containing 6 μl BSA; 400 ng ofend-labeled RSVhDAF plasmid were subsequently added to each sample for 2h; sperm cells were then washed and counted as described. (C) Lightmicroscope autoradiography of pig ejaculated sperm cells and nucleiafter 2 h incubation with ³H end-labeled RSVhDAF plasmid, Ejaculated andwashed sperm cells (panel a) and nuclei (panel b), ejaculated non washedsperm cells (panel c), ejaculated and washed sperm cells pre-incubatedwith 20 μl of seminal fluid (panel d). After DNA incubation, sperm cellsor nuclei were washed, spread on glass slides, and autoradiographed asdescribed. In successful experiments exogenous DNA is bound to thesub-equatorial region of sperm cells and nuclei (panels a and b). H&Estain, 1000× magnification.

FIG. 5 shows time course uptake of end-labeled RSVhDAF plasmid with pigsperm cells from 9 boars. End-labeled RSVhDAF plasmid was incubated at17° C. with ejaculated and washed sperm cells from the 9 pigs listed inTable 1. Uptake protocol as in FIG. 2.

FIG. 6 shows optimization of DNA uptake. Parallel time courseexperiments performed at different BSA and DNA concentrations, andtemperatures were performed to determine the best DNA incubationconditions for sperm cells from any given donor. In each experiment, oneparameter varied, while the remaining two were fixed. Determination ofuptake (in cpm) as in FIG. 4. (A) Parallel uptake experiments ofend-labeled RSVhDAF plasmid in sperm from boards Z, C, S, V. Each samplecontained 10⁶ sperm cells incubated with 400 ng of end-labeled RSVhDAFplasmid at 17° C. for 60 min in SFM plus different amounts of BSA (from6 to 30 g/l). (B) Parallel time course uptake experiments of end-labeledRSVhDAF plasmid in sperm from boar Z. Sperm cells were resuspended at aconcentration of 5×10⁶ cells/ml in SFM containing 6 g/l BSA, mixed withend-labeled RSVhDAF plasmid DNA (2 μg/ml) and incubated at differenttemperatures. Samples containing 106 sperm were withdrawn at theindicated times, washed and counted as in FIG. 4. (C) Parallel uptakeexperiments of end-labeled RSVhDAF plasmid in sperm from boards Z, C, S,V, L. For each sample 106 sperm cells in SFM containing 6 g/l BSA wereincubated at 17° C. for 60 min with increasing amounts of end-labeledRSVhDAF plasmid. (D) Light microscope autoradiography of ejaculated andwashed sperm cells from pig Z overloaded with end-labeled RSVhDAFplasmid.

DETAILED DESCRIPTION OF THE EMBODIMENTS

A. Media and Compositions

As described above, the inventors have found that the constitution ofthe medium in which sperm cells are processed during SMGT is importantfor improving efficiency of SMGT. Typically, the medium comprises, inwater, glucose in a concentration of about 56.19 to 68.67 mM, sodiumcitrate in a concentration of about 30.60 to 37.40 mM, EDTA in aconcentration of about 11.37 to 13.89 mM, citric acid in a concentrationof about 13.92 to 17.02 mM and Trizma base in a concentration of about48.31 to 59.05 mM. Typically the medium has an osmolarity of from about200 to 320 mOs. Typically the medium has a pH of about pH 7.4.

Typically, the concentration of glucose is about 62.43 mM, theconcentration of sodium citrate is about 34 mM, the concentration ofEDTA is about 12.6 mM, the concentration of citric acid is about 15.7 mMand the concentration of Trizma base is about 53.68 mM.

In one embodiment, the medium has an osmolarity of between 276 to 298mOs at pH 7.4. Preferably, the medium has an osmolarity of 286 mOs atpH7.4.

The inventors believe that glucose is important for providing a suitableosmolarity for the sperm cells, and for providing an energy source forsupporting the viability of the sperm cells. It follow that glucosecould be substituted for other sugars capable of providing thesefunctions. An example is fructose. The concentration of fructoserequired for supporting the viability of sperm cells can be determinedusing standard techniques known to the skilled addressee.

The inventors also believe that Trizma base (otherwise knowntromethamine as 2-amino-2-hydroxymethyl-1,3propanediol), and probablysodium citrate and citric acid, are important for providing a bufferingsystem for supporting a suitable pH for the sperm cells. In this regard,the medium is particularly suited to SMGT protocols that use in vivoartificial insemination. The medium is an advance over in vitrofertilization media such as TALP, because these media use a bufferingsystem that uses carbonate and use of a CO₂ incubator (both of which areused for in vitro fertilization). It follows that one of thesecomponents of the medium of the invention could be substituted foranother compound capable of providing a buffering system for supportingthe desired pH, provided that the compound is not toxic to sperm cells.

The inventors believe that EDTA is important for limiting the capacityof endonucleases to cleave the exogenous nucleic acid molecule to beused a transgene. EDTA affects endonuclease activity by chelatingcations, particularly calcium ions, so as to limit the availability ofthese ions. It is believed that EDTA may be substituted for anothercompound capable of providing this function, for example a chelator thatis not toxic to sperm cells.

Typically, the medium comprises a protein source for the sperm cells,such as bovine serum albumin. Bovine serum is typically in aconcentration of 6 g/litre of the medium. The inventors believe thatother sources of protein could be used, including, for example, otheralbumins or serum proteins.

Typically, the water for use in the medium is sterilised and purifiedsufficient to limit components in the medium that may cause injury tothe sperm cells. For example, the water may be double distilled orde-ionised to remove impurities and autoclaved to sterilise the water.

As described above, the invention also provides a process for theproduction of a medium for supporting the viability of a sperm cell.Typically, the process comprises contacting glucose in an amount ofabout 10.125 to 12.375 g, sodium citrate (2H₂O) in an amount of about9.00 to 11.00 g, EDTA (2H₂O) in an amount of about 4.23 to 5.17 g,citric acid (H₂O) in an amount of about 2.925 to 3.575 g, Trizma base inan amount of about 5.85 to 7.15 g, with about 1 litre of water, to forma solution. Typically the solution has a pH of about pH7.4 and anosmolarity ranging from about 200 to 320 mOs.

In one embodiment, glucose is in an amount of about 11.25 g, sodiumcitrate (2H₂O) is in an amount of about 10 g, EDTA (2H₂O) is in anamount of about 4.7 g, citric acid (H₂O) is in an amount of about 3.25g, and Trizma base is in an amount of about 6.5 g.

The protein source for the sperm cells may be added after the solutionhas been formed. Typically the protein source is added in an amount toprovide 6 g/litre of protein source. As described above, the proteinsource is typically bovine serum albumin, however, it is believed thatother sources could be used, including other serum albumins.

Where the medium is to be sterilised prior to use, particularly byautoclaving the solution, the protein source is added aftersterilisation and prior to use of the medium. This prevents denaturationof the protein source by the sterilising procedure.

The pH of the solution is typically adjusted to provide a pH of about7.4.

The invention also provides a medium for supporting the viability of asperm cell, the medium being produced by the above described process.

As described above, the invention also advantageously provides acomposition that can be used to provide a medium for supporting theviability of a sperm cell. The composition is preferably provided in asolid form and is adapted to provide the medium of the invention whendissolved in water. The composition comprises glucose, sodium citrate,citric acid EDTA and Trizma base in amounts sufficient for providing anaqueous solution having a concentration of glucose of from about 56.19to 68.67 mM, a concentration of sodium citrate of from about 30.60 to37.40 mM, a concentration of EDTA of from about 11.37 to 13.89 mM, aconcentration of citric acid of from about 13.92 to 17.02 and aconcentration of Trizma base of from about 48.31 to 59.05 mM.

Typically, the composition comprises glucose, sodium citrate, citricacid, EDTA and Trizma base in amounts sufficient for providing anaqueous solution having a concentration of glucose of about 62.43 mM, aconcentration of sodium citrate of about 34 mM, a concentration of EDTAof about 12.6 mM, a concentration of citric acid of about 15.7 mM and aconcentration of Trizma base of about 53.68 mM.

The composition may additionally contain a protein source for the spermcells, such as bovine serum albumin in an amount for providing theaqueous solution with a concentration of about 6 g/l of protein source.

In one embodiment, the composition comprises water. 11 this embodiment,the composition is advantageously provided as a concentrate to whichwater is to be added to provide a medium according to the first aspectof the invention.

B. Collection of a Sample of Sperm Cells

The invention also provides a method for collecting sperm cells from ananimal. An advantage of the method is that the sperm cells collectedaccording to the method are optimised for use in SMGT. As describedherein, the inventors have found that seminal fluid impinges on theefficiency of SMGT for transgenesis. While factors in seminal fluid havebeen hypothesised to effect DNA uptake in vitro [Zani et al. 1995 Exp.Cell Res. 217:57-64], the effect of these factors on the efficiency ofSMGT for transgenesis was not known prior to this invention.

According to the method, a sample of semen derived from an animal iscontacted with a medium according to the invention, to dilute the sampleof semen. It is believed that such dilution affects the capacity offactors in the seminal fluid to inhibit uptake of an exogenous nucleicacid molecule for use as a transgene. Accordingly, the frequency ofsperm cells transfected with the exogenous nucleic acid molecule isimproved.

It is preferable that the sample for collection is one that has beenfreshly ejaculated. Thus in one embodiment, the sample of semen is afreshly ejaculated sample.

The sample preferably comprises the first fraction of the ejaculatedsemen that represents about 30 to 40% of the total volume of theejaculated semen. This sample is believed to contain fewer factors forinhibiting uptake of a nucleic acid molecule. Thus in one embodiment,the sample comprises an initial 30 to 40% of the total volume of theejaculated semen.

One way of contacting the sample of semen with the medium is to firstpour the medium into a vessel and to then collect the sample of semeninto the vessel, so that the sample contacts the medium, to dilute thesample of semen in the medium. Thus in one embodiment, the sample ofsemen is collected into a vessel comprising the medium, to dilute thesample of semen. Alternatively, the sample of semen may be collectedinto a vessel and the medium subsequently added to the vessel to contactthe sample of semen and so dilute it.

The vessel and/or the medium may be pre-warmed to a temperature forsupporting the viability of a sperm cell, for example, they may bepre-warmed to about 37° C. This is useful for ensuring that theviability of the sperm cell is supported during collection. Thus in oneembodiment, the vessel and/or medium are pre-warmed to a temperature forsupporting the viability of a sperm cell, prior to contact of the mediumwith the sample of semen.

Typically the sample of semen is contacted with more or less an equalvolume of medium, to dilute the semen sample. The exact volume of mediumis not important, provided that it is sufficient for limiting contact ofsperm cells with factors in the seminal fluid that inhibit uptake of anucleic acid molecule by sperm cells. Thus in one embodiment, the volumeof medium contacted with the sample of semen is equal to the volume ofthe sample of semen.

C. Preparing Sperm Cells for Transfection

The invention comprises a method for preparing sperm cells fortransfection, or in other words, for uptake of a nucleic acid molecule.An advantage of the method is that the sperm cells prepared according tothe method are optimised for use in SMGT. The method comprises washing asperm cell in a medium according to the invention, to remove seminalfluid from the sperm cell. Where the sperm cells are to be used forSMGT, it is important to determine the motility of sperm cells in thesample, for example, as described below, because the motility of spermcells is directly related to a capacity to uptake a nucleic acidmolecule.

It is preferred that all seminal fluid is removed from the sperm cells,as this prevents factors in the seminal fluid from inhibiting uptake ofthe nucleic acid molecule. Accordingly, in one embodiment, all seminalfluid is removed from the sperm cell. It is recognised however, that itis not necessary that all seminal fluid be removed from the sperm cell,as long as the activity of factors in seminal fluid for inhibitinguptake of the nucleic acid molecule by the sperm cell is a leastlimited.

Typically, the sperm cell is washed according to the following steps:

-   -   (a) contacting a sample of semen derived from the animal with a        medium according to the first aspect of the invention, to dilute        the sample of semen;    -   (b) isolating sperm cells from the diluted sample;    -   (c) contacting the isolated sperm cells with a medium according        to the first aspect of the invention; and    -   (d) isolating sperm cells from the medium.

After step (a), the diluted sample of semen may be incubated at roomtemperature for about 5 minutes, before proceeding to step (b). Thisstep is useful because it provides an opportunity for equilibration andconditioning of the sperm cells in the medium.

It is not important how sperm cells are separated according to step (b),as long as the motility of the sperm cells is maintained. Preferably,the sperm cells are isolated in a first step by centrifugation.Exemplary conditions for centrifugation are about 800 g at about 25° C.for about 10 minutes. As described herein, these conditions limit lossof motility of sperm cells. Other conditions can be determined by theskilled addressee. In a second step, a supernatant that is formed by thesedimentation of sperm cells during the centrifugation is removed byaspiration to complete the isolation process of step (b).

According to step (c), the isolated sperm cells are contacted with amedium according to the first aspect of the invention. Typically, thevolume of medium is at least about 500 tines the volume of the pellet.It is preferable that the effect of the contact is to resuspend thesperm cells from the pellet. One way of resuspending cells is to gentlyflush the pellet with a wide-mouth pipette.

The isolation step of step (d) can be performed as described above inrelation to step (b).

Where the sperm cells prepared by the method are to be used for SMGT, itis important to determine the motility of sperm cells in the sample atthis stage. Methods for determining motility of sperm cells aredescribed further herein. Further, at least 1×10⁹ sperm cells arerequired for transfection, so after step (d), it is important that thesperm cells are suspended to a concentration suitable for this purpose.

D. Transfecting Sperm Cells

The invention provides a method for transfecting sperm cells, or inother words, for permitting sperm cells to uptake a nucleic acidmolecule. An advantage of the method is that the sperm cells preparedaccording to the method are optimised for use in SMGT. The methodcomprises contacting the sperm cell with the nucleic acid molecule in amedium according to the invention.

As seminal fluid contains factors that inhibit uptake of a nucleic acidmolecule by a sperm, the sperm cell is washed to remove seminal fluidbefore contact with the nucleic acid molecule. The method describedabove for washing a sperm cell is suitable for this purpose. Thus in oneembodiment, the sperm cell is free of seminal fluid before contact withthe nucleic acid molecule in the medium.

As described herein, the inventors have found that uptake of a nucleicacid molecule is optimised when 90% of the sperm cells bind to thenucleic acid molecule and 70% of these cells internalise the moleculeinto the nucleus. Further, approximately 20% of sperm bound nucleic acidmolecule is internalised into sperm nuclei. Thus in one embodiment, asample of sperm cells and the nucleic acid molecule are contacted in themedium of the invention in conditions for permitting about 90% of spermcells in the sample to bind to the nucleic acid molecule. In anotherembodiment, the sample of sperm cells and the nucleic acid molecule arecontacted in the medium of the invention in conditions for permittingabout 60% of the sperm cells to which nucleic acid molecule has bound,to internalise the nucleic acid molecule. In another embodiment, a spermcell and nucleic acid molecule are contacted in the medium in conditionsfor permitting about 20% of nucleic acid molecule bound to the spermcell to be internalised into the sperm cell nucleus.

The inventors have found that uptake of a nucleic acid molecule isoptimal where sperm cells and nucleic acid molecule are contacted in thefollowing amounts: about 1×10⁹ sperm cells with about 400 ug of nucleicacid molecule. The period of contact is typically about 2 to 4 hours andthe temperature is about 17 to 20° C.

The inventors have also found that the uptake of a nucleic acid moleculeis optimal where sperm is contacted with a nucleic acid molecule at anearly stage of capacitation. Where the sperm cell is prepared accordingto the methods described above in section C, this means that an idealtime for contact of the nucleic acid molecule with the sperm is within30 minutes after washing the sperm and no later than 60 minutes afterwashing the sperm. Methods for monitoring capacitation are describedherein.

E. SMGT—Artificial Insemination and In Vitro Fertilisation

SMGT can be performed using in vivo artificial insemination or in vitrofertilisation techniques. These techniques are known to the skilledaddressee. Methods for in vivo artificial insemination are describedherein, particularly in Standard Operating Procedure, Dept. Of NaturalResources and Environment, Victoria Government, Australia. In vivoartificial insemination techniques for SMGT are also described in[Sperandio et al. 1996 Animal Biotech. 7:58-77].

Methods for in vitro fertilisation are known to the skilled addresseeand are described in [Ball et al. 1983 Biol. Reprod. 28:717-725].

F. Producing Animals Comprising 2 or More Trangenes

As described above, the invention also provides a method of producing ananimal comprising 2 or more transgenes. An advantage of the method isthat many of the intercrosses of particular founders are not required toarrive an “ultimate” animal comprising the desired genotype, so that the“ultimate” animal can be generated with minimal time and expense. Themethod comprises the following steps:

-   -   (a) contacting a sperm cell with 2 or more exogenous nucleic        acid molecules, each for use as a transgene, to transfect the        sperm cell with each of the 2 or more exogenous nucleic acid        molecules;    -   (b) fertilising an ovum with the transfected sperm cell to        permit each of the 2 or more exogenous nucleic acid molecules to        be transferred to the ovum; and    -   (c) maintaining the fertilised ovum in conditions for permitting        the fertilised ovum to form the animal.

The method is important because it avoids the need for the intercrossesof particular founders that would otherwise be required to obtain thedesired genotype.

The sperm cell may be transfected by contacting the 2 or more exogenousnucleic acid molecules with the sperm cell in the medium of theinvention.

While not wanting to be bound by hypothesis, it is believed thatphysical linkage of the 2 or more exogenous nucleic acid molecules mayimpinge on the capacity of the method to produce an animal thatcomprises functional transgenes. That is, a consequence of physicallinkage may be rearrangement of the linked nucleic acid molecules whichwould destroy functionality of the transgene. Accordingly, the 2 or moreexogenous nucleic acid molecules are typically provided for transfectionas molecules that are not physically linked; in other words, the 2 ormore exogenous nucleic acid molecules are each discrete molecularentities.

Typically, in step (a), the two or more exogenous nucleic acid moleculesare contacted with the sperm cell to transfect the sperm cell at thesame time, or in other words, in the same incubation step. The inventorsrecognise that in some circumstances, it may be necessary to contact oneor more of the two or more nucleic acid molecules with the sperm cell inseparate steps. Thus in one embodiment, step (a) comprises contactingone or more of the two or more exogenous nucleic acid molecules with thesperm cell to transfect the sperm cell, and is proceeded by anadditional step of contacting the remaining molecules of the two or moreexogenous nucleic acid molecules with the sperm cell to transfect thesperm cell.

The sperm cell is contacted with the two or more exogenous nucleic acidmolecules in conditions for supporting the viability of the sperm cell.Examples of such conditions are described above in section D, and aredescribed further below in the Examples.

The sperm cells for use in the method may be collected according to themethods described above in section B and may be prepared according tothe methods described above in section C.

F. Cells, Tissues and Animals Derived from SMGT

As described above, in a seventh aspect, the invention provides spermcells transfected according to the method of the invention and in aneighth aspect, to cells tissues and animals prepared by fertilisation ofan ovum with said sperm cells.

Also in a further aspect, the invention provides cells, tissues andanimals characterised in that they are produced by SMGT and comprise twoor more transgenes.

Pigs and porcine cells and tissues are particularly preferred. Examplesof suitable pigs are described herein and include Landrace, Large Whiteor Landrace x Large White.

Examples of methods for collecting sperm cells are described above.Methods for collecting other cells and tissues are known to the skilledaddressee.

Typically, the cells, tissues and animals are homozygous for the one ormore transgenes. Homozygous cells and tissues can be obtained by anintercross of founders that are heterozygous for the transgene.

The following tissues and organs are particularly preferred: liver,heart, thyroid, adrenal, pancreas, pancreatic islets, kidney, bonemarrow, lymphocytes, neurons and lungs.

G. Selecting Donors for Providing Sperm Cells

As described above, the invention also provides methods of determiningwhether sperm cells are optimal for transfection and for use in SMGT.The inventors have found that the quality of sperm is an importantconsideration for optimisation of the efficiency of SMGT fortransgenesis. These methods comprise the step of determining whether atleast 65% of sperm cells in a sample are motile.

Also provided are methods for selecting sperm donors, for example boars,that are capable of producing sperm that are optimal for transfectionand for use in SMGT. These methods comprises the steps of determiningmotility of sperm cells in a sample and selecting a sample in which themotility is determined to be at least about 65%.

Typically, the higher % of motile sperm in the sample, the moreoptimised the sperm in the sample are for transfection and for SMGT.Preferably at least about 75% of sperm in the sample are motile. Morepreferably, at least about 85% of sperm in the sample are motile.

Typically, the motility of the sperm is determined after the sperm havebeen prepared according to the method described above in section C.Accordingly, in one embodiment, the method comprises determining whetherat least about 65% of sperm cells are motile in the medium of the firstaspect. of the invention. However, the inventors believe that therelationship between sperm motility and uptake of a nucleic acidmolecule is not dependent on the medium of the invention andaccordingly, the motility may be determined when the sperm cells arecomprised in another fertilization medium, such as TALP.

As described herein, where the sperm cells are to be used for SMGT,other characteristics of a sperm donor, i.e. characteristics in additionto sperm motility, may be considered. These include the breed and age ofthe donor and the fertility results of the sperm donor, includingconception rate and litter size. These characteristics are describedfurther herein. It will be recognised however, that the characteristicof sperm motility is most important in relation to optimising theefficiency of transfection of sperm cells and for optimising SMGT.

EXAMPLES Example 1 Preparation of Media

The medium for supporting the viability of a sperm cell (spermfertilization medium or SFM) was prepared as follows:

a. Reagents

-   -   All reagents were obtained from commercial sources as follows:    -   (D)+Glucose-anhydrous (Sigma Ultra) (Cat # G7528)    -   Sodium Citrate Trisodium salt: dihydrate (ASC reagent—Sigma)        (Cat#S4041)    -   Ethylenediaminetetractic acid Disodium Salt: dihydrate (Sigma        Ultra) (Cat#El644)    -   Citric acid monohydrate (Sigma Ultra) (Cat#C0706)    -   Trizma Base (Sigma Ultra) (Cat#T6791)    -   Bovine Albumin (BSA-Dried)—CSL (Cat#06711701)

b. Preparation of Media

SFM was prepared by forming a solution of 11.25 g (D)+Glucose-anhydrous,10 g Sodium Citrate Trisodium salt: dihydrate, 4.7 gEthylenediaminetetractic acid Disodium Salt: dihydrate, 3.25 g Citricacid monohydrate, 6.5 g Trizma Base in 1 litre of distilled autoclavedwater. The solution was adjusted to pH7.4 with 1N HCl and autoclaved.The osmolarity of the medium was about 286 mOs. Medium with mOs valuesbetween 250-300 or between 276-298 are suitable.

Bovine serum albumin was added to 6 g/L prior to use. The finalconcentration of BSA was varied for each sperm donor in accordance withoptimal DNA binding and uptake as described below.

Where artificial insemination is to be used for mediating gene transferinto an ovum, the SFM was prepared as a fresh solution one day beforeinsemination.

Example 2 Collecting a Sample of Sperm Cells from a Mammal

a Animals.

The breeds in this study were Landrace (sperm donors) and Large White orLandrace x Large White (gilts) swine.

All animals were housed and used in compliance with animal careguidelines.

b. Collection of Sperm

Briefly, semen was collected from the donor in a sterile plastic bagplaced in a thermostatic container pre-warmed at 37° C., to avoidtemperature shock. Quality of semen was evaluated on a slide pre-warmedat 37° C. Only the initial 30-40% of the ejaculate was collected sincethis fraction contains most of the sperm cells and a low amount ofseminal fluid, which may antagonise binding of DNA to sperm cells.

Example 3 Preparing a Collected Sample of Sperm Cells for Further Study

After collection of a sample of sperm cells, seminal fluid wassubsequently removed by carefully washing the sperm. Briefly, 5 mlaliquots of semen were transferred to 15 ml tubes and mixed with anequal volume of SFM supplemented with 6 mg/ml BSA pre-warmed at 37° C.(from this moment on the medium was kept at room temperature). Semen wasincubated for 5 min and then transferred to 50 ml tubes that were filledwith SFM/BSA to 50 ml. Samples were spun down at 800 g for 10 min at 25°C. and the supernatants were removed by aspiration without perturbingthe pellets, and discarded. The tubes were filled again with SFM/BSA,spun at 800 g for 10 min at 17 C and the supernatants discarded. Thesperm cells were carefully resuspended in the residual medium using awide tip-pipette and the pellets combined in one tube. Sperm cells werecounted using a hemocytometric chamber.

Example 4 Selecting a Donor for Providing a Sample of Sperm Cells

A sample of sperm cells was collected from each donor once every four tofive days on average according to Example 2 and prepared according toExample 3. Donors were selected for providing sperm cells for use inSMGT by assessment of semen quality and DNA binding.

a. Semen Quality

Sperm cells were collected once every four-five days on average, and thesemen quality evaluated by assessment of motility. Sperm motility wastested by microscopic inspection of semen on a slide pre-warmed to 37°C. Donors from which motility was observed in at least 80% of the totalsperm cells initially collected, and not less than 65% of sperm cellsafter preparing the collected sample according to Example 3 above, in atleast six ejaculates collected over a period of one month, were selectedfor further study.

Membrane integrity of sperm was measured by means of the hypo-osmoticswelling test [Oosterhuir GJ et al. 1996 J Clin Lab Anal. 10: 209-212].

b. DNA Binding

Sperm cells were prepared as described in Example 3. Following counting,the sperm was re-suspended at 1×10⁷ cells/mL, mixed with linearised,random prime ³H-deoxy-cytidine radio-labelled DNA (0.5 ug/mL) which was1-3×10⁶ CPM/1 g, and incubated at 18° C. Aliquots of sperm (1×10⁶) weretaken at specific times, diluted in Eppendorf tubes containing 1 mL SFM,washed twice by centrifuging at 4000 rpm for 5 minutes and re-suspendedin SFM (200 uL).

c. DNA in Nuclei

At the same time as an aliquot was taken for DNA binding analysis, 3×10⁶sperm were also taken and washed with SFM. Sperm were re-suspended in 26uL DTT buffer (DTT 100 mM, TRIS 50M, pH 7.5) and incubated on ice for 30minutes. {fraction (1/9)} of the volume (ie: 8.6 uL) of CTAB(cetyltrimethylamnonium bromide) buffer (CTAB 10%, DTT 10 mM) was added(1% v/v CTAB final) and incubated ⁴5 minutes on ice. Nuclei werepelleted at 900 rpm for 5′ at room temperature. Pellets are washed with500 uL of Lysis Buffer (20 mM Tris pH 7.5, EDTA 1 mM, NaCl 10 mM, 1%SDS) by centrifuging (9000 rpm for 5′ at room temperature). Pellets weredissolved in 100 uL 1M NaOH for at least 1 hour at 37° C. andneutralised with an equal volume of 1M HCl.

Radioactivity of samples was determined by liquid scintillation countsin 5 mL Packard Ultima Gold scintillation cocktail (Cat # 6013329). FIG.1 shows uptake of ³H-deoxy-cytidine radiolabelled linear EBFP plasmidDNA by pig sperm from 5 boars (a-e).

Autoradiography was also performed to verify that exogenous DNA weretaken up by the majority of the sperm cells and nuclei, and that DNAwere correctly localized within the sperms. Aliquots of ³H-radiolabelledsperm cells were washed and treated according to Lavitrano et al. 1992Mol. Reprod. Dev. 31: 161-169.

d. Sperm Capacitation

This was monitored by observing changes in motility and by using thechlorotetracycline fluorescence assay as described in Ward and StoreyDev Biol 1984 104: 287-296, and Barboni 1994 Zygote 2: 367-369.

Example 5 Methods of Transfecting Sperm Cells—Uptake and Expression ofMultiple Genes

Semen was collected from a transgenic CD46 boar according to Example 2and prepared as described in Example 3.

10⁹ viable sperm was diluted to 120 mL with 18° C. pre-equilibratedSFM/BSA 400 ug of DNA was added per 106 sperm cells. DNA consisted ofequal parts EBFP (enhanced blue fluorescent protein), EBFP, (enhancedgreen fluorescent protein) and dsRed2 plasmid DNA which had beenlinearised with EcoRI. The sperm and DNA were incubated at 18° C. for 2hour (optimal uptake time for the sperm from the boar used) with theflask gently inverted every 15-20 minutes.

Example 6 Methods of SMGT—Artificial Insemination

Sperm cells were transfected with a transgene according to Example 5,however in the final 20 minutes, the incubation was performed at roomtemperate and then 37° C. for the final minute before insemination.

A cycling sow was artificially inseminated using an inseminating pipetteand loading the incubate directly into the uterus according to standardprocedures (eg Standard Operating Procedure, Dept. Of Natural Resourcesand Environment, Victoria Government, Australia.

The sow was sacrificed at day 6 and embryos (predominantly late morula,early blastocyst stage) were harvested by flushing each uterine hornwith 30 mL Embryo Flushing Media (Dulbecco's phosphate buffer saline(PBS⁺) with 0.4% BSA w/v, 0.34 mM pyruvate and 5.5 mM glucose) at 36° C.Recovered embryos were examined by UV microscopy (Leica DMR) forexpression of the fluorescent DNA.

More specifically, the Living Colours Fluorescent Proteins haveexcitation maxima/emission maxima of 380 nm/440 nm, 488 nm/507 nm and558 nm/583 nm for EBFP, EGFP and dsRed2 respectively. The Lw microscopehas a green filter (Leica L9) which has an excitation of 450-490 nm andemission of 515-560 nm (bandpass) with dicot. mirror at 510. The redfilter (Leica N2.1) has an excitition of 515-560 nm and emission of 580nm (longpass) with a dicot. mirror of 480. The blue filter (Leica A4)has an excitation of 360 nm peak with a ½ band of 40 (ie: 360\40 nm) andemission of 470\40 nm with a dicot. mirror at 400. These filters havebeen shown to be specific for detection of each fluorescent protein bythe transfection of the CHOP cell line with each of the fluorescentproteins. Cells transfected with EBFP can only be detected in the bluefilter, cells transfected with EGFP can only be detected with the greenfilter and cells transfected with dsRed2 can only be detected with thered filter. The bleed through of green fluorescence into the red filterand red fluorescence into the green filter is negligable/absent usingthese filters on the UV microscope, as opposed to the FACS were there isleakage in the green and red channels requiring compensations to be set.

The presence of exogenous or transferred DNA in embryos was alsodetected by PCR, screening both the nucleotide sequence and the promoterusing two sets of primer pairs. Transgene expression was also examinedby Western blots. Screening of offspring is undertaken using PCR andSouthern blot analysis of DNA extracted from ear and tail tissues aftercleaning to remove potential contaminants.

Example 7 Production of Transgenic Embryos by SMGT

All 91 embryo's generated by fertilisation of eggs with semen which hadtaken up EBFP, EGFP and dsRed2 exogenous DNA expressed all three coloursof fluorescence proteins (blue, green and red).

In a preliminary experiment using only EGFP DNA, all 7 fertilisedembryos that were recovered expressed green fluorescent protein.

Control embryos which are fertilised with semen that has not beenincubated with exogenous DNA encoding for the fluorescence proteins donot express any fluorescence and serve as the negative control.

Maternal blood was taken from a CD46 sow at Day 98 followinginsemination. This sow had been inseminated with sperm from a CD46 boarwhich had taken up EBFP, EGEP and dsRed2 DNA and had been shown to bepregnant by oesterone sulphate assay of a Day 26 bleed (the standard wayof detecting pregnancy).

The PBMC fraction from the day 98 bleed contained cells and cell debriswhich expressed all three colours of fluorescence. This suggests that atleast one foetus of the pregnant sow expresses all three fluorescencegenes.

Example 8 Analysis of Optimised SMGT Efficiency

Materials and Methods

a Animals

The breeds in this study were Landrace (sperm donors) and Large White orLandrace x Large White (gilts) swine. Artificial inseminations wereperformed using DNA loaded sperm, prepared as described below, fromselected boars kept trained and not exposed to natural mating.Prepubertal gilts (animals that had never given birth), weighing 70-80kg, were synchronised by injecting 1250 IU of ECG (Folligon, Intervet,Holland) and 60 h later 750 IU of hCG (Corulon, Intervet, Holland).Ovulation occured 40 h after hCG injection. Artificial inseminationswere performed 43 h after hCG injection using 1-1.5×10⁹ DNA treatedsperm cells/gilt. Artificial insemination was then carried out with aninseminating pipette according to standard procedures.

All animals were housed and used in compliance with animal careguidelines.

b. Preparation of Sperm, DNA Uptake and Artificial Insemination

Samples of sperm cells were collected according to Example 2 andprepared according to Example 3 above.

10⁹ sperm cells were incubated with 400 μg plasmid DNA at 17° C. inSFM/BSA for 2 h. Artificial insemination is carried out using aninseminating pipette according to standard procedures.

c. Assessment of DNA Uptake.

Scintillation counting was performed on ejaculated or ejaculated andwashed sperm cells resuspended at a concentration of 5.10⁶ cells/ml inSFM, containing 6 g/l BSA, mixed with ³H end-labelled RSVhDAF plasmidDAN (2 μg/ml) and incubated at 17° C., all unless specified differently.Aliquots containing 1.×10⁶ sperm cells were withdrawn from theincubation mixture at specific times, diluted in Eppendorf tubes,containing 1 ml of SFM and washed twice by centrifuging at 4000 rpm for5 min in a microfuge. In order to prepare nuclei, aliquots containing atleast 3.10⁶ sperm cells were withdrawn at the same time points andthoroughly washed with SFM. Nuclei were prepared, briefly, as follows:sperm cells were suspended in DTT buffer (DTT 100 mM, Tris 50 mM pH 7.5)at the concentration of 1×10⁶ sperm cells/26 μl and incubated for 30 minon ice; CTAB (Cetyltrimethylammonium bromide, Sigma Aldrich, St. Louis,Mo.) solution (CTAB 10%, DTT 10 mM) is added at {fraction (1/9)} thevolume (1% CTAB final) and further incubated for 45-60 min on ice.Nuclei were pelleted at 9000 rpm in a microfuge for 5 min at roomtemperature and pellets are washed with 500 μl of 50 mM Tris pH 8.0 bycentrifuging as above. Sperm or nuclear pellets were dissolved in 100 μlof 1 M NaOH for at 14 est 1 h at 37° C., neutralized with an equalvolume of 1 M HCl, and counted. For autoradiography, aliquots of spermcells were incubated with ³H-end-labeled plasmid with a specificactivity of 1×10⁶ cpm/1 g, washed and treated. For both types of assays,scintillation counting and autoradiography, DNA end-labeling is carriedout by filling in the 5′ protruding ends of the linearized plasmid usingthe Klenow subunit of DNA polymerase I.

d. Preparation of Seminal Fluid and DNA Uptake Inhibition

Seminal fluid was prepared by centrifugation of pig semen. Sperm cellswere sedimented at 700 g for 10 mins. Supernatants were furthercentrifuged for 1 min at 12000 g in a microfuge. Increasing amounts ofseminal fluid were mixed with ejaculated washed sperm cells in a volumeof 200 μl containing 1×10⁶ sperm cells and incubated for 30 min at 17°C. Labeled DNA (400 ng/200 μl) was then added for an additional 60 min.Washing, counting and autoradiography were as described above.

Results

a. Technical Aspects of the SMGT Method in Pigs

We have established the parameters that optimise efficiency of SMGT. Weused as test gene of interest a minigene for human decay acceleratingfactor (hDAF), expression of which has been shown to help prolongsurvival of pig organs in non-human primates.

b. Selection of Appropriate Sperm Donors and Optimization of Sperm/DNAInteraction are the Key Steps for Successful SMGT

Choice of a good sperm donor requires significant effort, however, oncea boar is chosen, that boar can be used for years, infact sperm cellsfrom a given boar show highly reproducible characteristics over time.

Selection of sperm donors requires evaluating (i) the quality of semenbased on standard parameters used in conventional animal breedingprograms and (ii) the ability of the sperm cells to take up andinternalize exogenous DNA (FIG. 4).

Sperm quality is influenced by many factors, such as the season of theyear (semen quality declines significantly during the hot season),collection frequency, breed and age of the donor. The breed used in thisstudy was Landrace (3-4 year old boars), because the semen quality isbetter than in Large White or Duroc. Sperm collection in our study wasno more frequent than every four days; the boars normally ejaculatedevery four to five days. Sperm quality was evaluated by fertilityresults (conception rate and litter size) obtained at breeding farms[Holt et al. 1997 J. Androl. 18:312-323]. In addition, we paidparticular attention to high progressive motility of sperm. To testmotility, microscopic inspection of the semen was performed on a slideprewarmed to 37° C. Table 1 shows the results for 9 of the 20 boardstested. Motility should be at least 85% initially, and not less than 65%after the washing procedure, on at least six different ejaculatescollected over a period of one month. Membrane integrity of the spermcells is documented by means of the hypo-osmotic swelling test [Austin1952 Nature 170:326; Oosterhuis et al. 1996 J. Clin. Lab. Anal.10:209-212].

A critical parameter in sperm selection is the ability of sperm cells tobind exogenous DNA and internalize it into their nuclei. DNA uptakecorrelates with semen quality, particularly in terms of “highprogressive motility” of the sperm after ejaculation. DNA uptake isassessed by two techniques. The amount of labelled exogenous DNA boundto sperm cells or nuclei is determined by scintillation counting in timecourse experiments. Autoradiographic experiments are also performed foreach time point to verify that the exogenous DNA is correctly localizedwithin sperm cells or nuclei (see Materials and Methods). FIG. 4A showsa time course of uptake of end-labeled RSVhDAF plasmid by ejaculated pigsperm cells and its internalization into nuclei. We found that untreatedejaculated pig spermatozoa do not take up exogenous DNA (FIG. 4A, ▴);DNA uptake takes place only after extensive washing of the sperm and“complete” removal of seminal fluid as shown in FIG. 4A (▪).Approximately 20% of the DNA bound to the sperm is internalized into thesperm nuclei (“FIG. 4A, ●). Autoradiographic analysis ofejaculated-washed sperm cells incubated for 2 h with DNA shows that thebinding occurs on the sub-acrosomal region of the sperm head and spermnuclei (FIG. 4, panel C a and b). In the non-washed ejaculated spermcells there is essentially no binding of exogenous DNA (FIG. 4, panelCc). Furthermore, small amounts of seminal fluid added to thoroughlywashed sperm prevents DNA uptake (FIG. 4, panels B and Cd).

A number of donors chosen on the basis of semen quality were screenedfor DNA uptake. FIG. 5 shows a time course of DNA uptake by sperm cellsfrom the 9 boars described in Table 1. Sperm cells from the 9 boarsreproducibly differ greatly in their capacity to take up exogenous DNA,although the kinetics of uptake is similar. In all cases there is rapidbinding of most of the DNA during the initial 15-30 min followed after60 min by a plateau. DNA uptake correlates with semen quality,particularly in terms of high progressive motility (Table 1). Two boars,Z and S, were selected and used in the SMGT expeliments. The boars thatwere chosen are thus the ones that took up comparatively high amounts ofDNA, with the DNA contained in most of the sperm and properly localized.

c. Optimization of DNA Uptake

In acceptable experiments, exogenous DNA binds to about 90% of the spermcells and binding is followed by nuclear internalization in 70% of thosecells. (FIG. 6 Panels A and C). We found: in general that approximately20% of the sperm-bound DNA is internalized into sperm nuclei. Nuclearinternalization is completed within 60 min (FIG. 4 panels A and C b). Tooptimize the protocol for generating transgenic animals, it is necessaryto establish when, for how long and in what quantity DNA must be addedto sperm. These parameters, discussed below, were assessed by loadingwashed sperm cells and end-labeled DNA at various times aftercollection, and assessing DNA uptake.

d. When is the Best Time for Initiating Sperm-DNA Interaction?

There is a window of opportunity that coincides with the early stage ofcapacitation. DNA is ideally added within 30 min after washing the spermand not later than 60 min. Capacitation is the time during which anumber of physiological changes take place that make sperm competent tofertilize (Austin 1952). Capacitation was monitored by observing changesin motility and by carrying out the chlortetracycline (CTC) fluorescence(Ward et al. 1984; Barboni 1994). Chlortetracycline staining allows fora rapid evaluation of sperm capacitation, showing different fluorescentpatterns according to the different functional status of sperm.Capacitation time should be modulated (see below) so as to allow forcomplete interaction between the sperm and the DNA.

e. How Long Should Sperm be Incubated with Exogenous DNA?

Acceptable sperm donors should be able to complete DNA-sperm interactionwithin 2 to 4 h, during which time capacitation must be allowed toproceed at its normal pace, avoiding acceleration of the process. Thepresence of calcium in the medium promotes the likelihood thatendogenous endonucleases will cleave exogenous DNA, potentially leadingto integration of rearranged DNA and triggering of apoptotic events inthe sperm genome. We thus developed a calcium-free medium. Using thiscalcium-free medium, which slows down capacitation time after removal ofseminal fluid, we varied the temperature and the amount of BSA added tothe medium, which modulate capacitation time. We have carried out anumber of time course experiments aimed at optimizing the uptake processin the absence of calcium. Parallel time course experiments at differenttemperatures (17° C., 20° C., 25° C., 37° C.) or BSA concentrations(from 6 to 30 g/l) were performed to determine the best DNA incubationconditions for sperm cells from any given donor (FIGS. 6A and B). Suchexperiments must be carried out on the sperm of each selected boar. Inthe experiments reported in FIG. 6A, varying amounts of BSA were addedto the calcium-free medium. Sperm preparations from boars Z, S and C areall acceptable, based on their responses to the different concentrationsof BSA, however, S and C are best if incubated with 6 μl, whereas Zappears to perform better by this assay when incubated with at 30 μl.However, at the highest concentration of BSA, the DNA did not localizecorrectly in the sperm of Z, i.e., the sperm were overloaded with DNA,as shown by autoradiographic studies (FIG. 6D). Thus for Z as well weused 6 μl BSA in SFM. Sperm from boar V was rejected, given the very lowuptake, which is also evidenced by the results of autoradiography.

f. At What Temperature Should the DNA be Added to the Sperm?

In general, we found that incubation at 17° C. to 20° C. was best forDNA uptake; at this temperature there is diminished nuclease activity ascompared to higher temperatures and decreased motility, thus energy isconserved for artificial insemination (FIG. 6B).

g. How Much DNA Should be Added?

It is important to optimize the amount of exogenous DNA per sperm cell,so as to obtain the highest number of sperm containing DNA withoutoverloading (FIG. 6D). DNA-overloaded sperms could be damaged ordisadvantaged in fertilization compared with normal spermatozoa, andartificial insemination could amplify the disadvantage. An importantparameter in this aspect of testing is whether sperm is resistant toincreasing concentrations of DNA, i.e., whether overloading will lead todecreased uptake. Sperm from boar Z again performs best by this assay.Whereas C and S both seemed acceptable when testing concentrations ofBSA added, the valuation of added DNA showed that S was resistant tohigher concentrations (the uptake did not decrease), while C was not(uptake decreased with DNA concentrations above 400 ng/1×10⁶ sperm).Thus, S was chosen as a sperm donor while C was not (FIG. 6C).

h. Using the SMGT Method: Production of Transgenic Pigs by SpermMediated Gene Transfer

Sperm cells from two selected boars (Z and S) were used as vectors fortransferring into eggs by artificial insemination a 6.8 kb constructcontaining a hDAF minigene. Fifteen gilts were fertilized in eightexperiments over a period of eighteen months. 93 piglets were generated.Table 1 summarises DNA and RNA studies based on PCR, Southern blot,RT-PCR and Northern blot analyses. Southern blots were performed on DNAextracted from tail and ear tissue biopsies collected at birth, Northernblots and RT-PCR on RNA samples from ear, liver or muscle biopsies frompits positive for DNA. DNA of 53 of the 93 animals (57%) contained hDAFsequences. The HDAR transgene was transcribed in all tissues tested in34/53 (64%) of the animals. The expressed gene product was stablytransmitted to progeny, found in caveolae as it is in human cells, andfunctional in several assays.

1. A medium for supporting the viability of a sperm cell comprising, in water, glucose in a concentration of about 56 to 69 mM, sodium citrate in a concentration of about 31 to 37 mM, EDTA in a concentration of about 11 to 14 mM, citric acid in a concentration of about 14 to 17 mM and Trizma base in a concentration of about 48 to 59 mM, wherein the medium has an osmolarity of from about 200 to 320 mOs and a pH of about pH 7.4.
 2. A medium according to claim 1 wherein the concentration of glucose is from about 56.19 to 68.67 mM, the concentration of sodium citrate is from about 30.60 to 37.40 mM, the concentration of EDTA is from about 11.37 to 13.89 mM, the concentration of citric acid is from about 13.92 to 17.02 mM and the concentration of Trizma base is from about 48.31 to 59.05 mM.
 3. A medium according to claim 2 wherein the concentration of glucose is about 62.43 mM, the concentration of sodium citrate is about 34 mM, the concentration of EDTA is about 12.6 mM, the concentration of citric acid is about 15.7 mM and the concentration of Trizma base is about 53.68 mM.
 4. A medium according to claim 1 wherein the medium has an osmolarity of between 276 to 298 mOs at pH 7.4.
 5. A medium according to claim 4 wherein the medium has an osmolarity of 286 mOs at pH7.4.
 6. A medium according to claim 1, further comprising 6 g/liter of bovine serum albumin.
 7. A process for the production of a medium for supporting the viability of a sperm cell comprising contacting glucose in an amount of about 10.1 to 12.4 g, sodium citrate (2H₂O) in an amount of about 9.0 to 11.0 g, EDTA (2H₂O) in an amount of about 4.2 to 5.2 g, citric acid (H₂O) in an amount of about 2.9 to 3.6 g and Trizma base in an amount of about 5.9 to 7.2 g, with about 1 liter of water, to form a solution with a pH of about pH7.4 and an osmolarity ranging from about 200 to 320 mOs.
 8. A process according to claim 7 wherein the amount of glucose is from about 10.125 to 12.375 g, the amount of sodium citrate (2H₂O) is from about 9.00 to 11.00 g, the amount of EDTA (2H₂O) is from about 4.23 to 5.17 g, the amount of citric acid (H₂O) is from about 2.925 to 3.575 g, the amount of Trizma base is from about 5.85 to 7.15 g.
 9. A process according to claim 8 wherein glucose is in an amount of about 11.25 g, sodium citrate (2H₂O) is in an amount of about 109, EDTA (2H₂O) is in an amount of about 4.7 g, citric acid (H₂O) is in an amount of about 3.25 g, and Trizma base is in an amount of about 6.5 g.
 10. A process according to claim 7 wherein the pH of the solution is adjusted to provide a pH of about 7.4.
 11. A process according to claim 7, further comprising contacting the solution with 6 g bovine serum albumin per liter of solution.
 12. A medium for supporting the viability of a sperm cell, the medium being produced by the process according to claim
 7. 13. A composition for providing a medium for supporting the viability of a sperm cell comprising glucose, sodium citrate, citric acid, EDTA and Trizma base in amounts sufficient for providing an aqueous solution having a concentration of glucose of from about 56 to 69 mM, a concentration of sodium citrate of from about 31 to 37 mM, a concentration of EDTA of from about 11 to 14 mM, a concentration of citric acid of from about 14 to 17 mM and a concentration of Trizma base of from about 48 to 59 mM.
 14. A composition according to claim 13 wherein glucose, sodium citrate, citric acid, EDTA and Trizma base are in amounts sufficient for providing an aqueous solution having a concentration of glucose of from about 56.19 to 68.67 mM, a concentration of sodium citrate of from about 60 to 37.40 mM, a concentration of EDTA of from about 11.37 to 13.89 mM, a concentration of citric acid of from about 13.92 to 17.02 mM and a concentration of Trizma base of from about 48.31 to 59.05 mM.
 15. A composition according to claim 14 wherein glucose, sodium citrate, citric acid, EDTA and Trizma base are comprised in amounts sufficient for providing an aqueous solution having a concentration of glucose of about 62.43 mM, a concentration of sodium citrate of about 34 mM, a concentration of EDTA of about 12.6 mM, a concentration of citric acid of about 15.7 mM and a concentration of Trizma base of about 53.68 mM.
 16. A composition according to claim 13 further comprising bovine serum albumin in an amount for providing an aqueous solution with a concentration of about 6 g/l of bovine serum albumin.
 17. A composition according to claim 13 wherein the composition comprises water.
 18. A method for collecting sperm cells from an animal for use in sperm-mediated gene transfer comprising contacting a semen sample derived from the animal with a medium according to claim 1, to dilute the sample of semen.
 19. A method according to claim 18 wherein the sample of semen is a freshly ejaculated sample.
 20. A method according to claim 19 wherein the sample comprises an initial 30 to 40% of the total volume of the ejaculated semen.
 21. A method according to claim 18 wherein the sample of semen is collected into a vessel comprising the medium, to dilute the sample of semen.
 22. A method according to claim 21 wherein at least one of the vessel and/or medium are pre-warmed to a temperature for supporting the viability of a sperm cell, prior to contact of the medium with the sample of semen.
 23. A method according to claim 18 wherein a volume of medium contacted with the sample of semen is equal to the volume of the sample of semen.
 24. A method for preparing a sperm cell for use in sperm-mediated gene transfer comprising washing a sperm cell in a medium according to claim 1, to remove seminal fluid from the sperm cell.
 25. A method according to claim 24 wherein all seminal fluid is removed from the sperm cell.
 26. A method according to claim 24 wherein the sperm cell is washed according to the following steps: (a) contacting a sample of semen derived from the animal with a medium according to claim 1, to dilute the sample of semen; (b) isolating sperm cells from the diluted sample; (c) contacting the isolated sperm cells with a medium according to claim 1; and (d) isolating sperm cells from the medium.
 27. A method for transfecting a sperm cell with a nucleic acid molecule comprising contacting the sperm cell with the nucleic acid molecule in a medium according to claim
 1. 28. A method according to claim 27 wherein the sperm cell and nucleic acid molecule are contacted in the medium in conditions for permitting about 90% of the sperm cells to bind to the nucleic acid molecule.
 29. A method according to claim 28 wherein the sperm cell and nucleic acid molecule are contacted in the medium in conditions for permitting about 60% of the sperm cells to which nucleic acid molecule is bound to internalized the nucleic acid molecule.
 30. A method according to claim 27 wherein the sperm cell and nucleic acid molecule are contacted in the medium in conditions for permitting about 20% of nucleic acid molecule bound to a sperm cell to be internalized into the sperm cell nucleus.
 31. A method according to claim 27 wherein about 1×109 sperm cells are contacted with about 400 ug of nucleic acid molecule.
 32. A method according to claim 27 wherein the sperm cell and nucleic acid molecule are contacted in the medium for about 2 to 4 hours at about 17 to 20° C.
 33. A sperm cell transfected according to the method of claim
 27. 34. A cell prepared by fertilization of an ovum with a sperm according to claim
 33. 35. A method for determining whether a sample of sperm cells is optimal for transfection comprising determining whether at least about 65% of the sperm cells in the sample are motile.
 36. A method for selecting a sample of sperm cells for transfection comprising: (a) determining the motility of sperm cells in a sample; and (b) selecting a sample in which the motility of sperm cells is determined to be at least about 65%.
 37. A method for determining whether a sample of sperm cells are optimal for introducing a transgene into an oocyte comprising determining whether at least about 65% of sperm cells in the sample are motile.
 38. A method for selecting a sample of sperm cells that are optimized for introducing a transgene into an oocyte comprising: (a) determining the motility of sperm cells in a sample; and (b) selecting a sample in which the motility of sperm cells is determined to be at least about 65%.
 39. A method of producing an animal comprising at least 2 transgenes comprising: (a) contacting a sperm cell with at least 2 exogenous nucleic acid molecules, each for use as a transgene, to transfect the sperm cell with each of the at least 2 exogenous nucleic acid molecules; (b) fertilizing an ovum with the transfected sperm cell to permit each of the at least 2 exogenous nucleic acid molecules to be transferred to the ovum; and (c) maintaining the fertilized ovum in conditions for permitting the fertilized ovum to form the animal.
 40. A non-human animal produced by the method of claim
 39. 